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Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital
JIPH-453; No. of Pages 8
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Journal of Infection and Public Health (2015) xxx, xxx—xxx
SHORT COMMUNICATION
Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital Dipendra Thapaliya a,b,1, Ashley M. O’Brien a,b, Shylo E. Wardyn a,b, Tara C. Smith a,b,∗,1 a
Department of Epidemiology, University of Iowa College of Public Health, Iowa City, IA, USA b Center for Emerging Infectious Diseases, University of Iowa College of Public Health, Iowa City, IA, USA Received 2 February 2015 ; received in revised form 28 May 2015; accepted 12 June 2015
KEYWORDS Staphylococcus aureus; Streptococcus pyogenes; Necrotizing fasciitis; Epidemiology; Infection
Summary The present study was performed to characterize the epidemiology of necrotizing soft tissue infection caused by Streptococcus pyogenes (n = 14) and Staphylococcus aureus (n = 14) isolates collected at the University of Iowa Hospitals and Clinics. An additional 9 S. pyogenes isolates were collected from patients being treated for mild respiratory infections and served as a comparison sample in the analysis. Patient data corresponding to the isolates (n = 37) were also collected in order to identify risk factors or comorbid conditions possibly correlated with necrotizing fasciitis (NF). The prevalence of methicillin-resistant S. aureus among the study isolates was 35.7% (5/14), and the prevalence of the Panton—Valentine leukocidin (PVL) gene was 57% (8/14). The S. pyogenes NF (wound) isolates (n = 14) belonged to 10 different emm types, none of which appeared to be associated with more severe disease when compared to the milder infection (throat) samples (n = 9). Comorbid conditions such as diabetes and cardiovascular disease were significantly associated with NF. The results indicate that there may be a high prevalence of the PVL virulence factor in NF infections and that spa type t008 may be responsible for the increasing incidence of S. aureus NF infections in Iowa. © 2015 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences.
∗
Corresponding author at: 750 Hilltop Drive, Lowry Hall, Kent, OH 44242, USA. Tel.: +1 330 672 3946. E-mail address: [email protected] (T.C. Smith). 1 Present address: Department of Biostatistics, Environmental Health Sciences, & Epidemiology, College of Public Health, Kent State University, Kent, OH, USA. http://dx.doi.org/10.1016/j.jiph.2015.06.003 1876-0341/© 2015 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences.
Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
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D. Thapaliya et al.
Introduction
Materials and methods
In the United States, the incidence of necrotizing infections is approximately 0.04 cases per 1000 persons-years [1]; nevertheless, the disease is often characterized by its rapid progression and high mortality rate, which is estimated to range from 25% to 35% [1,2], though some studies have reported a mortality rate as high as 50% [3]. Most necrotizing infection cases are caused by Streptococcus pyogenes; however, the incidence of necrotizing fasciitis (NF) with Staphylococcus aureus identified as the primary pathogen is on the rise [4,5]. To date, little is known about the pathogenicity of necrotizing infections, primarily regarding molecular characterization and virulence factors, as well as host factors that may be correlated with severe necrotizing infections. Three notable virulence factors associated with S. pyogenes infection include emm type, mutations in the covR/covS system, and the presence of superantigens. Previous research has indicated that certain emm types may be responsible for necrotizing S. pyogenes infections, particularly emm types 1, 3, and 12 [6—8]. A key virulence factor of interest for S. aureus infections is the presence of the Panton—Valentine leukocidin (PVL) gene. Of particular interest, two previous studies performed molecular typing on subsets of methicillin-resistant S. aureus (MRSA) isolates causing NF and reported the presence of the PVL gene in 100% of the samples (5/5) [4,9]. Along with molecular characterization, numerous studies have investigated the association between certain host factors and an increased risk of NF. Studies examining patient medical histories have identified a multitude of factors, including prior trauma, surgery, nonsteroidal antiinflammatory drug use, burns, chronic alcohol consumption, immunosuppressive drug use, cancer, diabetes, obesity, and renal disease, among others, that may be correlated with NF [10—12]. However, studies also have reported the occurrence of necrotizing infections in patients with no known risk factors or comorbid conditions [4,11]. The previously described findings demonstrate the need for further research regarding the etiology of NF. The goals of our study were to determine the molecular characterization of NF caused by S. pyogenes and S. aureus, including emm type (S. pyogenes), spa type, and the presence of the PVL and mecA genes (S. aureus), and to identify whether specific host factors are correlated with severe necrotizing infections among patients at the University of Iowa Hospitals and Clinics.
Study isolates (n = 38) were collected and stored at the University of Iowa Hospitals and Clinics Pathology Department between January 2011 and September 2012. Fourteen S. aureus and 14 S. pyogenes samples were collected from cases of necrotizing infection; the remaining 10 S. pyogenes isolates were obtained from throat cultures collected from patients being treated for mild respiratory infections and served as a comparison in the analysis. Isolates were attained from the Pathology Department in October 2012 following IRB approval, and were analyzed at the Center for Emerging Infectious Diseases with molecular typing. Genomic DNA extraction was performed using the Wizard Genomic DNA preparation kit (Promega, WI). Polymerase chain reaction (PCR) was performed to detect mecA and PVL genes (lukS, lukF) present in the S. aureus isolates [13,14]. The staphylococcus protein A (spa) gene was amplified using SpaF (5 -GAACAA-CGTAACGGCTTCATCC-3 ) and 1514R (5 -CAGCAGTAGTGCCGTTTGCCT-3 ), as previously described [15,16]; emm typing was carried out for all S. pyogenes isolates [17], and 16s rRNA PCR was performed with all isolates to confirm the species [18]. Upon completion of 16s rRNA PCR, 1 isolate among the S. pyogenes throat samples was found to belong to the Streptococcus parasanguinis species, and was subsequently excluded from further analysis, leaving 9 throat infection isolates. Multilocus sequence typing was completed on all but 1 study isolate [19]. All S. aureus and 23 S. pyogenes isolates were tested for antibiotic susceptibility by using the VITEK 2 System (bioMérieux). We used the ASTGP71 and AST-ST01 cards of the VITEK 2 System for the antibiotic susceptibility testing of S. aureus and S. pyogenes, respectively. S. aureus isolates were tested for susceptibility to benzylpenicillin, oxacillin, tetracycline, erythromycin, ciprofloxacin, moxifloxacin, minocycline, clindamycin, quinupristin/ trimethoprim—sulfamethoxazole, dalfopristin, gentamicin, levofloxacin, linezolid, daptomycin, vancomycin, rifampicin, minocycline, tigecycline, and nitrofurantoin. S. pyogenes isolates were tested for susceptibility to benzylpenicillin, ampicillin, cefotaxime, ceftriaxone, tetracycline, erythromycin, clindamycin, levofloxacin, trimethoprim—sulfamethoxazole, linezolid, and vancomycin. Isolates showing intermediate levels of susceptibility were classified as resistant. S. aureus isolates that were resistant to 3 or more classes of antimicrobials or that were
Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
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resistant to oxacillin were considered multidrug resistant [20]. We retrieved patient data from the medical records of the University of Iowa Hospitals and Clinics and linked patients’ medical record numbers to the bacterial specimens for the purposes of investigating host factors that may be associated with necrotizing infection. Patients’ demographics, clinical data, comorbid conditions, outcome variables, and specimen-specific data were extracted and entered into the database. Analysis of patient data was conducted using SAS statistical software (version 9.3, SAS Institute Inc., Cary, NC).
Results Molecular typing revealed 9 different spa types among the S. aureus study isolates, with the only repeat type being t008 (43%; n = 6). The prevalence of MRSA in the study sample was 35.7% (5/14). All of the MRSA isolates were spa type t008. Fifty-seven percent (8/14) of the S. aureus isolates harbored the PVL gene, while all MRSA isolates harbored the PVL genes. Most of the S. aureus isolates (86%; 12/14) contained a functional agr. A total of 7 different sequence types were detected among 14 necrotizing infection S. aureus isolates. Among S. aureus isolates, ST8 was the most common sequence type identified (46.1%; 6/13), followed by ST188 (15.4%; 2/13). No other sequence types (38.5%; 5/13) were found to be repeated (Table 1a). Among the S. pyogenes isolates, there were 14 different emm types represented: 17.4% were emm12 (n = 4), 13% were emm11 (n = 3), 13% were emm89 (n = 3), and 8.7% were emm77 (n = 2) (Table 1b). A total of 15 different sequence types Table 1a
were detected among the 14 necrotizing infection and 9 throat infection S. pyogenes isolates. ST36 was the most common sequence type identified (17%; 4/23), followed by ST678 (13%; 3/23), ST101 (13%; 3/23), and ST63 (9%; 2/23). No other sequence types (48%; 11/23) were found to be repeated. Among 14 S. aureus isolates, oxacillin resistance was observed in 35.7% (5/14). Four isolates (28.6%) were resistant to ciprofloxacin; 7 (50%) were resistant to erythromycin; 2 (14.3%) were resistant to clindamycin; 3 (21.4%) were resistant to levofloxacin; and all 14 (100%) were resistant to benzylpenicillin. Eight isolates (57%) were multidrug resistant S. aureus. Among 23 S. pyogenes isolates, 7 isolates (30.4%) were resistant to erythromycin; 1 (4.3%) was resistant to ciprofloxacin; 8 (34.8%) were resistant to clindamycin; 1 (4.3%) was resistant to ceftriaxone; 7 (30.4%) were resistant to tetracycline; and 1 (4.3%) was resistant to cefotaxime. Sixty-nine percent (9/13) and 31% (4/13) of the NF patients were male and female, respectively. Sixty-nine percent of the patients (9/13) were white, and 31% (4/13) were African American. The median age of the patients was 48 years (n = 13; mean, 42.69; standard deviation, 12.65; range, 21—61) (Table 2). The median length of hospitalization was 10 days (n = 13; mean, 12.4; standard deviation, 10.2; range, 1—38 days). The majority of patients (92%; 12/13) diagnosed with NF underwent surgery. The sites of NF included the extremity (left foot: 2; right foot: 1; left ankle: 1; left lower leg: 1; right lower leg: 2) in 7 patients (53.8%), trunk (left upper back and back trunk) in 2 patients (15.4%), perineum in 3 patients (23.1%), and both trunk and extremity in 1 patient (7.7%) (Table 2).
Molecular characteristics of S. aureus.
Isolate ID
Source
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing
infection infection infection infection infection infection infection infection infection infection infection infection infection infection
mecA
PVL
spa
AST
MLST
agr phenotype
− − − − − − + − + + + − + −
− − − + − − + − + + + + + +
t189 t1109 t701 t008 t088 t8870 t008 t5051 t008 t008 t008 t094 t008 t10075
P, P, P, P, P P P P P, P, P, P P, P,
ST188 ST97 ST6 ST8 ST5 ST188 ST8 ST45 ST8 ST8 ST8 ST15 ST8 ST152
Functional Functional Functional Functional Functional Functional Functional Functional Functional Functional Functional Dysfunctional Functional Dysfunctional
C, E, Cl O, C, L, E E, Cl E
T, TS O O, C, L, E O, C, L, E O, E
Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
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D. Thapaliya et al. Table 1b
Molecular characteristics of S. pyogenes.
Isolate ID
Source
EMM type
AST
MLST
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection
emm1.0 emm6.4 emm12.0 emm87.0 emm102.2 emm28.4 emm92.0 emm12.0 emm103.0 emm11.0 emm2.0 emm89.0 st106M.5 emm89.0 emm3.1 emm89.0 emm12.0 emm77.0 emm11.0 emm77.0 emm4.0 emm12.0 emm11.0
— — — — C, CT, E, Cl — E, Cl, T E, Cl CT, CX E, Cl, T — — T — Cl — — T E, Cl, T T — E, Cl E, Cl, T
ST28 ST382 ST36 ST62 ST60 ST52 ST82 ST36 ST327 ST678 ST55 ST101 ST53 ST101 ST15 ST101 ST36 ST63 ST678 ST63 ST39 ST36 ST678
P, benzylpenicillin; C, ciprofloxacin; E, erythromycin; Cl, clindamycin; O, oxacillin; T, tetracycline; TS, trimethoprim/sulfamethoxazole; CT, ceftriaxone; CX, cefotaxime; —, susceptible to all antibiotics tested.
A majority of the patients (84.6%; 11/13) had at least 1 comorbid condition or risk factor; the most common comorbid condition was obesity (69.2%, 9/13), followed by cardiovascular disease (61.5%; 8/13), diabetes (61.5%; 8/13), pulmonary disease (30.8%; 4/13), neurological disorders (peripheral neuropathy, schizophrenia, anxiety, depression, panic attack, attention-deficit hyperactive disorder) (23%; 3/13), cancer (15.4%; 2/13), malnutrition (7.7%; 1/13), and illicit drug use (7.7%; 1/13). Two patients (15.4%; 2/13) had no serious comorbid conditions or risk factors (Table 2). There was a higher incidence of NF among men (69.2%; 9/13) compared to women (30.7%; 4/13). Similarly, white patients were more affected (69.2%; 9/13) than African—American patients (30.7%; 4/13). No other race or ethnicity was reported. Sixty-nine percent (9/13) and 31% (4/13) of the patients had monomicrobial and polymicrobial infections, respectively (Table 2).
Discussion More than 100 distinct molecular types of S. pyogenes have been identified on the basis of M protein, which is encoded by the emm gene [21]. It is hypothesized that some emm types may be more
pathogenic than others [22]. In this study, only 2 emm types (emm1.0 and emm87.0) were found in NF cases. All other NF cases diagnosed in this study were caused by S. aureus. In recent decades, the incidence of lifethreatening invasive infection of communityassociated MRSA (CA-MRSA) is increasing in many parts of the world [4]. MRSA has been identified as the most commonly isolated pathogen from patients with skin and soft tissue infections presenting in U.S. emergency departments [23]. In our study, 38.5% (5/13) of the NF infections were caused by CA-MRSA. The results of our study are consistent with a nationwide surveillance study that found a high prevalence of MRSA (51%; 2093/4131) in clinically significant S. aureus isolates [24]. In their study, USA300 was the most common strain (n = 1269; 61%), and two-thirds of USA300 isolates originated from wound and skin infections. Sixtyfour percent of the MRSA isolates harbored PVL genes [24]. This is in contrast to a study of S. aureus asymptomatic carriage conducted in the same area of Iowa as the current study, in which only 5.2% of isolates collected were positive for PVL [25], suggesting that invasive infections are enriched for PVL-positive isolates. Another study conducted in Los Angeles, California, reported 29% (9/31) of cases of NF were caused by CA-MRSA [4].
Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
Age
Sex
Race or ethnicity
Organisms in positive culture
Microbial status
Diagnosis
Duration of hospitalization (days)
Surgery
Site of infection
Comorbid conditions
1 & 15 2
49 31
F M
SA SA
p M
NF CL
16 2
Yes No
Left lower leg Lower abdomen
DM, CVD, OBS None
3
61
M
White American Indian African American
SA & SP
P
NF
38
Yes
Left foot
4
57
M
White
SA
M
NFb
6
Yes
Right foot
5
51
M
White
SA
M
NF
1
No
Perineal
6
44
M
White
SA
M
OST
5
Yes
Right foot
7
51
F
African American
SA
M
NF
15
Yes
Left upper back
8 9 10 11
52 32 21 23
M F F
White White White White
SA SA (MRSA) SA SA (MRSA)
P M M M
NF NF NF NF
4 25 7 13
Yes Yes Yes Yes
12
35
M
White
SA
M
NF
17
Yes
Right lower leg Perineal Left ankle Trunk & left upper arm Right foot
CVD, DM, PD, peripheral neuropathy, OBS CVD, DM, cancer, OBS CVD, DM, PD, OBS illicit drug use CVD, DM, PD, GID, peripheral neuropathy CVD, DM, PD, OBS, Schizophrenia CVD OBS None DM
13 14
36 48
M M
SA (MRSA) SA (MRSA)a
M P
NF NF
4 10
Yes Yes
Right lower leg Back trunk
16
32
M
White African American White
SP
M
CL
1
Yes
Right forearm
17
51
F
White
SP
M
BA
1
Yes
Left breast
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Characteristics of the patients.
Necrotizing fasciitis in Iowa
CVD, DM, PD, cancer, OBS, malnutrition, panic attack OBS CVD, DM, OBS Illicit drug use, depression, anxiety, ADHD None
5
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Table 2
Site of infection
Comorbid conditions
5
Yes
Perineal
None
AP & I
1
No
None
M
PRT
5
Yes
Neck (pharyngitis)/extremities (impetigo) Left hip
SP
M
1
No
P
3
No
Neck & right lower leg Left ankle
None
White White
SA (MRSA) & SP SP SP
CRB & AP CL
M M
CL SI
1 1
No No
M M
White White
SA & SP SA & SP
P P
CL PVD
5 1
Yes Yes
Perirectal Back/left shoulder & arm Left forearm Left foot
F F
White White
SP SP
M M
LCR CL
1 1
No No
Right hand Left elbow
Age
Sex
Race or ethnicity
Organisms in positive culture
Microbial status
Diagnosis
18
39
M
SP
M
NF
19
21
F
African American Hispanic/ multiracial
SP
M
20
39
M
SP
21
21
M
NonHispanic/ multiracial Unknown
22
44
M
White
23 24
23 41
M M
25 26
44 56
27 28
48 38
Duration of hospitalization (days)
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Surgery
Isolate ID
None
OBS None GID GID, ADHD CVD, malnutrition, GID, PTSD, depression None GID
D. Thapaliya et al.
Abbreviations used: SP, Streptococcus pyogenes; SA, Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; M, mono-microbial; P, poly-microbial; NF, necrotizing fasciitis; CL, cellulitis; OST, osteomyelitis; BA, breast abscess; AP, acute pharyngitis; FVR, fever/fatigue; SI, skin infection; I, impetigo; LCR, hand laceration; PRT, peritonitis; DM, diabetes mellitus, CVD, cardiovascular disease; PD, pulmonary disease; GID, gastrointestinal disease; ADHD, attention deficit hyperactivity disorder; PTSD, post-traumatic stress disorder. a Klebsiella pneumonia was also isolated from wound. b Secondary to diabetic cellulitis.
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Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
Table 2 (Continued)
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Necrotizing fasciitis in Iowa In contrast to the previous report of high prevalence of monomicrobial MRSA in wound cultures (86%; 12/14), only 3 cases of NF (23%; 3/13) in our study were caused by monomicrobial CA-MRSA [4]. However, consistent with a previous study in which all of the tested isolates (n = 5) were ST 8- and PVL-positive [4], ST8 (USA300/t008) was the most common sequence type in our study, and more than half of our S. aureus isolates and all MRSA isolates harbored the PVL gene. Although CA-MRSA USA300 has been well documented in many outbreaks of soft tissue infections, the findings of this study, those of another study that documented 16.7% of CA-MRSA NF cases were USA 300 [9], and those of a study by Miller et al. [4] demonstrate the rising incidence of NF caused by CA-MRSA in the United States. The typical mortality rate of NF is about 33% [4]. However, similar to a previous study [4], none of the NF patients in our study died; this may be due to differences in care, in study population, or in the bacteriology of the NF infections in this hospital. This finding also suggests the possibility that the strain of CA-MRSA causing NF in our study population may be less virulent than NF caused by other organisms. Previous studies have documented several risk factors for necrotizing soft tissue infection, including diabetes mellitus, intravenous drug abuse, peripheral vascular disease, obesity, malnutrition, blunt or penetrating trauma, alcohol abuse, surgical incisions, chicken pox, vesicles, and immunosuppression [1,2]. The majority of the patients of this study had at least 1 comorbid condition or risk factor. The S. pyogenes study isolates belonged to 14 different emm types, with no specific emm type being over-represented among the necrotizing infection cases versus the mild respiratory infection controls; however, no broad conclusions can be drawn due to the small sample size of NF cases caused by S. pyogenes. The results of this study indicate that several spa and emm types are responsible for NF cases presenting at the University of Iowa Hospitals and Clinics. All of the MRSA NF isolates in the study were spa type t008, suggesting this strain of S. aureus may be associated with increased disease severity in Iowa. Our study has several limitations. Though this study was prospective in design [11], not all microbial cultures were banked for later analysis. As such, our study included convenience samples of both S. aureus and S. pyogenes in 1 hospital, thus limiting the generalizability of results. However, this study provides insights into the molecular characteristics of S. aureus and S. pyogenes causing NF at University of Iowa Hospitals and Clinics. Future research should focus on analyzing samples
7 from all NF cases within a facility, and analyzing molecular characteristics of all causative agents.
Funding Center for Emerging Infectious Diseases, University of Iowa.
Conflict of interest None declared.
Ethical approval The University of Iowa IRB evaluated this project and determined that it did not qualify as human subjects research.
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Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
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Journal of Infection and Public Health (2015) xxx, xxx—xxx
SHORT COMMUNICATION
Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital Dipendra Thapaliya a,b,1, Ashley M. O’Brien a,b, Shylo E. Wardyn a,b, Tara C. Smith a,b,∗,1 a
Department of Epidemiology, University of Iowa College of Public Health, Iowa City, IA, USA b Center for Emerging Infectious Diseases, University of Iowa College of Public Health, Iowa City, IA, USA Received 2 February 2015 ; received in revised form 28 May 2015; accepted 12 June 2015
KEYWORDS Staphylococcus aureus; Streptococcus pyogenes; Necrotizing fasciitis; Epidemiology; Infection
Summary The present study was performed to characterize the epidemiology of necrotizing soft tissue infection caused by Streptococcus pyogenes (n = 14) and Staphylococcus aureus (n = 14) isolates collected at the University of Iowa Hospitals and Clinics. An additional 9 S. pyogenes isolates were collected from patients being treated for mild respiratory infections and served as a comparison sample in the analysis. Patient data corresponding to the isolates (n = 37) were also collected in order to identify risk factors or comorbid conditions possibly correlated with necrotizing fasciitis (NF). The prevalence of methicillin-resistant S. aureus among the study isolates was 35.7% (5/14), and the prevalence of the Panton—Valentine leukocidin (PVL) gene was 57% (8/14). The S. pyogenes NF (wound) isolates (n = 14) belonged to 10 different emm types, none of which appeared to be associated with more severe disease when compared to the milder infection (throat) samples (n = 9). Comorbid conditions such as diabetes and cardiovascular disease were significantly associated with NF. The results indicate that there may be a high prevalence of the PVL virulence factor in NF infections and that spa type t008 may be responsible for the increasing incidence of S. aureus NF infections in Iowa. © 2015 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences.
∗
Corresponding author at: 750 Hilltop Drive, Lowry Hall, Kent, OH 44242, USA. Tel.: +1 330 672 3946. E-mail address: [email protected] (T.C. Smith). 1 Present address: Department of Biostatistics, Environmental Health Sciences, & Epidemiology, College of Public Health, Kent State University, Kent, OH, USA. http://dx.doi.org/10.1016/j.jiph.2015.06.003 1876-0341/© 2015 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences.
Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
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Introduction
Materials and methods
In the United States, the incidence of necrotizing infections is approximately 0.04 cases per 1000 persons-years [1]; nevertheless, the disease is often characterized by its rapid progression and high mortality rate, which is estimated to range from 25% to 35% [1,2], though some studies have reported a mortality rate as high as 50% [3]. Most necrotizing infection cases are caused by Streptococcus pyogenes; however, the incidence of necrotizing fasciitis (NF) with Staphylococcus aureus identified as the primary pathogen is on the rise [4,5]. To date, little is known about the pathogenicity of necrotizing infections, primarily regarding molecular characterization and virulence factors, as well as host factors that may be correlated with severe necrotizing infections. Three notable virulence factors associated with S. pyogenes infection include emm type, mutations in the covR/covS system, and the presence of superantigens. Previous research has indicated that certain emm types may be responsible for necrotizing S. pyogenes infections, particularly emm types 1, 3, and 12 [6—8]. A key virulence factor of interest for S. aureus infections is the presence of the Panton—Valentine leukocidin (PVL) gene. Of particular interest, two previous studies performed molecular typing on subsets of methicillin-resistant S. aureus (MRSA) isolates causing NF and reported the presence of the PVL gene in 100% of the samples (5/5) [4,9]. Along with molecular characterization, numerous studies have investigated the association between certain host factors and an increased risk of NF. Studies examining patient medical histories have identified a multitude of factors, including prior trauma, surgery, nonsteroidal antiinflammatory drug use, burns, chronic alcohol consumption, immunosuppressive drug use, cancer, diabetes, obesity, and renal disease, among others, that may be correlated with NF [10—12]. However, studies also have reported the occurrence of necrotizing infections in patients with no known risk factors or comorbid conditions [4,11]. The previously described findings demonstrate the need for further research regarding the etiology of NF. The goals of our study were to determine the molecular characterization of NF caused by S. pyogenes and S. aureus, including emm type (S. pyogenes), spa type, and the presence of the PVL and mecA genes (S. aureus), and to identify whether specific host factors are correlated with severe necrotizing infections among patients at the University of Iowa Hospitals and Clinics.
Study isolates (n = 38) were collected and stored at the University of Iowa Hospitals and Clinics Pathology Department between January 2011 and September 2012. Fourteen S. aureus and 14 S. pyogenes samples were collected from cases of necrotizing infection; the remaining 10 S. pyogenes isolates were obtained from throat cultures collected from patients being treated for mild respiratory infections and served as a comparison in the analysis. Isolates were attained from the Pathology Department in October 2012 following IRB approval, and were analyzed at the Center for Emerging Infectious Diseases with molecular typing. Genomic DNA extraction was performed using the Wizard Genomic DNA preparation kit (Promega, WI). Polymerase chain reaction (PCR) was performed to detect mecA and PVL genes (lukS, lukF) present in the S. aureus isolates [13,14]. The staphylococcus protein A (spa) gene was amplified using SpaF (5 -GAACAA-CGTAACGGCTTCATCC-3 ) and 1514R (5 -CAGCAGTAGTGCCGTTTGCCT-3 ), as previously described [15,16]; emm typing was carried out for all S. pyogenes isolates [17], and 16s rRNA PCR was performed with all isolates to confirm the species [18]. Upon completion of 16s rRNA PCR, 1 isolate among the S. pyogenes throat samples was found to belong to the Streptococcus parasanguinis species, and was subsequently excluded from further analysis, leaving 9 throat infection isolates. Multilocus sequence typing was completed on all but 1 study isolate [19]. All S. aureus and 23 S. pyogenes isolates were tested for antibiotic susceptibility by using the VITEK 2 System (bioMérieux). We used the ASTGP71 and AST-ST01 cards of the VITEK 2 System for the antibiotic susceptibility testing of S. aureus and S. pyogenes, respectively. S. aureus isolates were tested for susceptibility to benzylpenicillin, oxacillin, tetracycline, erythromycin, ciprofloxacin, moxifloxacin, minocycline, clindamycin, quinupristin/ trimethoprim—sulfamethoxazole, dalfopristin, gentamicin, levofloxacin, linezolid, daptomycin, vancomycin, rifampicin, minocycline, tigecycline, and nitrofurantoin. S. pyogenes isolates were tested for susceptibility to benzylpenicillin, ampicillin, cefotaxime, ceftriaxone, tetracycline, erythromycin, clindamycin, levofloxacin, trimethoprim—sulfamethoxazole, linezolid, and vancomycin. Isolates showing intermediate levels of susceptibility were classified as resistant. S. aureus isolates that were resistant to 3 or more classes of antimicrobials or that were
Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
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resistant to oxacillin were considered multidrug resistant [20]. We retrieved patient data from the medical records of the University of Iowa Hospitals and Clinics and linked patients’ medical record numbers to the bacterial specimens for the purposes of investigating host factors that may be associated with necrotizing infection. Patients’ demographics, clinical data, comorbid conditions, outcome variables, and specimen-specific data were extracted and entered into the database. Analysis of patient data was conducted using SAS statistical software (version 9.3, SAS Institute Inc., Cary, NC).
Results Molecular typing revealed 9 different spa types among the S. aureus study isolates, with the only repeat type being t008 (43%; n = 6). The prevalence of MRSA in the study sample was 35.7% (5/14). All of the MRSA isolates were spa type t008. Fifty-seven percent (8/14) of the S. aureus isolates harbored the PVL gene, while all MRSA isolates harbored the PVL genes. Most of the S. aureus isolates (86%; 12/14) contained a functional agr. A total of 7 different sequence types were detected among 14 necrotizing infection S. aureus isolates. Among S. aureus isolates, ST8 was the most common sequence type identified (46.1%; 6/13), followed by ST188 (15.4%; 2/13). No other sequence types (38.5%; 5/13) were found to be repeated (Table 1a). Among the S. pyogenes isolates, there were 14 different emm types represented: 17.4% were emm12 (n = 4), 13% were emm11 (n = 3), 13% were emm89 (n = 3), and 8.7% were emm77 (n = 2) (Table 1b). A total of 15 different sequence types Table 1a
were detected among the 14 necrotizing infection and 9 throat infection S. pyogenes isolates. ST36 was the most common sequence type identified (17%; 4/23), followed by ST678 (13%; 3/23), ST101 (13%; 3/23), and ST63 (9%; 2/23). No other sequence types (48%; 11/23) were found to be repeated. Among 14 S. aureus isolates, oxacillin resistance was observed in 35.7% (5/14). Four isolates (28.6%) were resistant to ciprofloxacin; 7 (50%) were resistant to erythromycin; 2 (14.3%) were resistant to clindamycin; 3 (21.4%) were resistant to levofloxacin; and all 14 (100%) were resistant to benzylpenicillin. Eight isolates (57%) were multidrug resistant S. aureus. Among 23 S. pyogenes isolates, 7 isolates (30.4%) were resistant to erythromycin; 1 (4.3%) was resistant to ciprofloxacin; 8 (34.8%) were resistant to clindamycin; 1 (4.3%) was resistant to ceftriaxone; 7 (30.4%) were resistant to tetracycline; and 1 (4.3%) was resistant to cefotaxime. Sixty-nine percent (9/13) and 31% (4/13) of the NF patients were male and female, respectively. Sixty-nine percent of the patients (9/13) were white, and 31% (4/13) were African American. The median age of the patients was 48 years (n = 13; mean, 42.69; standard deviation, 12.65; range, 21—61) (Table 2). The median length of hospitalization was 10 days (n = 13; mean, 12.4; standard deviation, 10.2; range, 1—38 days). The majority of patients (92%; 12/13) diagnosed with NF underwent surgery. The sites of NF included the extremity (left foot: 2; right foot: 1; left ankle: 1; left lower leg: 1; right lower leg: 2) in 7 patients (53.8%), trunk (left upper back and back trunk) in 2 patients (15.4%), perineum in 3 patients (23.1%), and both trunk and extremity in 1 patient (7.7%) (Table 2).
Molecular characteristics of S. aureus.
Isolate ID
Source
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing Necrotizing
infection infection infection infection infection infection infection infection infection infection infection infection infection infection
mecA
PVL
spa
AST
MLST
agr phenotype
− − − − − − + − + + + − + −
− − − + − − + − + + + + + +
t189 t1109 t701 t008 t088 t8870 t008 t5051 t008 t008 t008 t094 t008 t10075
P, P, P, P, P P P P P, P, P, P P, P,
ST188 ST97 ST6 ST8 ST5 ST188 ST8 ST45 ST8 ST8 ST8 ST15 ST8 ST152
Functional Functional Functional Functional Functional Functional Functional Functional Functional Functional Functional Dysfunctional Functional Dysfunctional
C, E, Cl O, C, L, E E, Cl E
T, TS O O, C, L, E O, C, L, E O, E
Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
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D. Thapaliya et al. Table 1b
Molecular characteristics of S. pyogenes.
Isolate ID
Source
EMM type
AST
MLST
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Necrotizing infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection Respiratory infection
emm1.0 emm6.4 emm12.0 emm87.0 emm102.2 emm28.4 emm92.0 emm12.0 emm103.0 emm11.0 emm2.0 emm89.0 st106M.5 emm89.0 emm3.1 emm89.0 emm12.0 emm77.0 emm11.0 emm77.0 emm4.0 emm12.0 emm11.0
— — — — C, CT, E, Cl — E, Cl, T E, Cl CT, CX E, Cl, T — — T — Cl — — T E, Cl, T T — E, Cl E, Cl, T
ST28 ST382 ST36 ST62 ST60 ST52 ST82 ST36 ST327 ST678 ST55 ST101 ST53 ST101 ST15 ST101 ST36 ST63 ST678 ST63 ST39 ST36 ST678
P, benzylpenicillin; C, ciprofloxacin; E, erythromycin; Cl, clindamycin; O, oxacillin; T, tetracycline; TS, trimethoprim/sulfamethoxazole; CT, ceftriaxone; CX, cefotaxime; —, susceptible to all antibiotics tested.
A majority of the patients (84.6%; 11/13) had at least 1 comorbid condition or risk factor; the most common comorbid condition was obesity (69.2%, 9/13), followed by cardiovascular disease (61.5%; 8/13), diabetes (61.5%; 8/13), pulmonary disease (30.8%; 4/13), neurological disorders (peripheral neuropathy, schizophrenia, anxiety, depression, panic attack, attention-deficit hyperactive disorder) (23%; 3/13), cancer (15.4%; 2/13), malnutrition (7.7%; 1/13), and illicit drug use (7.7%; 1/13). Two patients (15.4%; 2/13) had no serious comorbid conditions or risk factors (Table 2). There was a higher incidence of NF among men (69.2%; 9/13) compared to women (30.7%; 4/13). Similarly, white patients were more affected (69.2%; 9/13) than African—American patients (30.7%; 4/13). No other race or ethnicity was reported. Sixty-nine percent (9/13) and 31% (4/13) of the patients had monomicrobial and polymicrobial infections, respectively (Table 2).
Discussion More than 100 distinct molecular types of S. pyogenes have been identified on the basis of M protein, which is encoded by the emm gene [21]. It is hypothesized that some emm types may be more
pathogenic than others [22]. In this study, only 2 emm types (emm1.0 and emm87.0) were found in NF cases. All other NF cases diagnosed in this study were caused by S. aureus. In recent decades, the incidence of lifethreatening invasive infection of communityassociated MRSA (CA-MRSA) is increasing in many parts of the world [4]. MRSA has been identified as the most commonly isolated pathogen from patients with skin and soft tissue infections presenting in U.S. emergency departments [23]. In our study, 38.5% (5/13) of the NF infections were caused by CA-MRSA. The results of our study are consistent with a nationwide surveillance study that found a high prevalence of MRSA (51%; 2093/4131) in clinically significant S. aureus isolates [24]. In their study, USA300 was the most common strain (n = 1269; 61%), and two-thirds of USA300 isolates originated from wound and skin infections. Sixtyfour percent of the MRSA isolates harbored PVL genes [24]. This is in contrast to a study of S. aureus asymptomatic carriage conducted in the same area of Iowa as the current study, in which only 5.2% of isolates collected were positive for PVL [25], suggesting that invasive infections are enriched for PVL-positive isolates. Another study conducted in Los Angeles, California, reported 29% (9/31) of cases of NF were caused by CA-MRSA [4].
Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
Age
Sex
Race or ethnicity
Organisms in positive culture
Microbial status
Diagnosis
Duration of hospitalization (days)
Surgery
Site of infection
Comorbid conditions
1 & 15 2
49 31
F M
SA SA
p M
NF CL
16 2
Yes No
Left lower leg Lower abdomen
DM, CVD, OBS None
3
61
M
White American Indian African American
SA & SP
P
NF
38
Yes
Left foot
4
57
M
White
SA
M
NFb
6
Yes
Right foot
5
51
M
White
SA
M
NF
1
No
Perineal
6
44
M
White
SA
M
OST
5
Yes
Right foot
7
51
F
African American
SA
M
NF
15
Yes
Left upper back
8 9 10 11
52 32 21 23
M F F
White White White White
SA SA (MRSA) SA SA (MRSA)
P M M M
NF NF NF NF
4 25 7 13
Yes Yes Yes Yes
12
35
M
White
SA
M
NF
17
Yes
Right lower leg Perineal Left ankle Trunk & left upper arm Right foot
CVD, DM, PD, peripheral neuropathy, OBS CVD, DM, cancer, OBS CVD, DM, PD, OBS illicit drug use CVD, DM, PD, GID, peripheral neuropathy CVD, DM, PD, OBS, Schizophrenia CVD OBS None DM
13 14
36 48
M M
SA (MRSA) SA (MRSA)a
M P
NF NF
4 10
Yes Yes
Right lower leg Back trunk
16
32
M
White African American White
SP
M
CL
1
Yes
Right forearm
17
51
F
White
SP
M
BA
1
Yes
Left breast
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Characteristics of the patients.
Necrotizing fasciitis in Iowa
CVD, DM, PD, cancer, OBS, malnutrition, panic attack OBS CVD, DM, OBS Illicit drug use, depression, anxiety, ADHD None
5
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Table 2
Site of infection
Comorbid conditions
5
Yes
Perineal
None
AP & I
1
No
None
M
PRT
5
Yes
Neck (pharyngitis)/extremities (impetigo) Left hip
SP
M
1
No
P
3
No
Neck & right lower leg Left ankle
None
White White
SA (MRSA) & SP SP SP
CRB & AP CL
M M
CL SI
1 1
No No
M M
White White
SA & SP SA & SP
P P
CL PVD
5 1
Yes Yes
Perirectal Back/left shoulder & arm Left forearm Left foot
F F
White White
SP SP
M M
LCR CL
1 1
No No
Right hand Left elbow
Age
Sex
Race or ethnicity
Organisms in positive culture
Microbial status
Diagnosis
18
39
M
SP
M
NF
19
21
F
African American Hispanic/ multiracial
SP
M
20
39
M
SP
21
21
M
NonHispanic/ multiracial Unknown
22
44
M
White
23 24
23 41
M M
25 26
44 56
27 28
48 38
Duration of hospitalization (days)
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Surgery
Isolate ID
None
OBS None GID GID, ADHD CVD, malnutrition, GID, PTSD, depression None GID
D. Thapaliya et al.
Abbreviations used: SP, Streptococcus pyogenes; SA, Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; M, mono-microbial; P, poly-microbial; NF, necrotizing fasciitis; CL, cellulitis; OST, osteomyelitis; BA, breast abscess; AP, acute pharyngitis; FVR, fever/fatigue; SI, skin infection; I, impetigo; LCR, hand laceration; PRT, peritonitis; DM, diabetes mellitus, CVD, cardiovascular disease; PD, pulmonary disease; GID, gastrointestinal disease; ADHD, attention deficit hyperactivity disorder; PTSD, post-traumatic stress disorder. a Klebsiella pneumonia was also isolated from wound. b Secondary to diabetic cellulitis.
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Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003
Table 2 (Continued)
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Necrotizing fasciitis in Iowa In contrast to the previous report of high prevalence of monomicrobial MRSA in wound cultures (86%; 12/14), only 3 cases of NF (23%; 3/13) in our study were caused by monomicrobial CA-MRSA [4]. However, consistent with a previous study in which all of the tested isolates (n = 5) were ST 8- and PVL-positive [4], ST8 (USA300/t008) was the most common sequence type in our study, and more than half of our S. aureus isolates and all MRSA isolates harbored the PVL gene. Although CA-MRSA USA300 has been well documented in many outbreaks of soft tissue infections, the findings of this study, those of another study that documented 16.7% of CA-MRSA NF cases were USA 300 [9], and those of a study by Miller et al. [4] demonstrate the rising incidence of NF caused by CA-MRSA in the United States. The typical mortality rate of NF is about 33% [4]. However, similar to a previous study [4], none of the NF patients in our study died; this may be due to differences in care, in study population, or in the bacteriology of the NF infections in this hospital. This finding also suggests the possibility that the strain of CA-MRSA causing NF in our study population may be less virulent than NF caused by other organisms. Previous studies have documented several risk factors for necrotizing soft tissue infection, including diabetes mellitus, intravenous drug abuse, peripheral vascular disease, obesity, malnutrition, blunt or penetrating trauma, alcohol abuse, surgical incisions, chicken pox, vesicles, and immunosuppression [1,2]. The majority of the patients of this study had at least 1 comorbid condition or risk factor. The S. pyogenes study isolates belonged to 14 different emm types, with no specific emm type being over-represented among the necrotizing infection cases versus the mild respiratory infection controls; however, no broad conclusions can be drawn due to the small sample size of NF cases caused by S. pyogenes. The results of this study indicate that several spa and emm types are responsible for NF cases presenting at the University of Iowa Hospitals and Clinics. All of the MRSA NF isolates in the study were spa type t008, suggesting this strain of S. aureus may be associated with increased disease severity in Iowa. Our study has several limitations. Though this study was prospective in design [11], not all microbial cultures were banked for later analysis. As such, our study included convenience samples of both S. aureus and S. pyogenes in 1 hospital, thus limiting the generalizability of results. However, this study provides insights into the molecular characteristics of S. aureus and S. pyogenes causing NF at University of Iowa Hospitals and Clinics. Future research should focus on analyzing samples
7 from all NF cases within a facility, and analyzing molecular characteristics of all causative agents.
Funding Center for Emerging Infectious Diseases, University of Iowa.
Conflict of interest None declared.
Ethical approval The University of Iowa IRB evaluated this project and determined that it did not qualify as human subjects research.
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Please cite this article in press as: Thapaliya D, et al. Epidemiology of necrotizing infection caused by Staphylococcus aureus and Streptococcus pyogenes at an Iowa hospital. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.06.003